The effects of neoadjuvant therapy on the methylation levels and gene expression of BCAT1 and IKZF1 in colorectal tumour and para-tumour tissues

Author: Maher Jedi

Jedi, Maher, 2016 The effects of neoadjuvant therapy on the methylation levels and gene expression of BCAT1 and IKZF1 in colorectal tumour and para-tumour tissues, Flinders University, School of Medicine

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Globally, colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third in males, with 1.4 million new cases estimated to have occurred in 2012. This disease arises as a consequence of several genetic and epigenetic modifications, including DNA methylation. Blood tests are being developed to detect these methylation changes for CRC screening. One such test is based on the presence of circulating hypermethylated BCAT1 and IKZF1, but little work has been done to measure methylation levels in the colorectal tumour and non-tumour tissue, or to determine the impact that methylation may have on the gene expression levels. In addition, it is not known how treatment (radiation/chemotherapy) of the tumour may affect methylation and gene expression. The aim of this study was to assess levels of methylation and gene expression of BCAT1 and IKZF1 in tumour and para-tumour tissues of CRC patients and to determine the effect of treatment on these levels. Tumour and adjacent non-tumour (para-tumour) tissues were collected at surgical resection of 50 patients diagnosed with different stages of CRC. Of these patients, 14 were subjected to treatment prior to surgical resection. DNA was extracted, bisulfite converted and levels of methylated BCAT1 and IKZF1 were evaluated via quantitative PCR and expressed as a percentage of β-actin levels. The relative gene expression levels of BCAT1 and IKZF1 were assessed through measuring mRNA levels by first extracting RNA, preparing cDNA and then performing real-time PCR to measure expression relative to a reference gene (HPRT1). Comparisons between the different groups were performed with a Mann Whitney U-test (p values <0.05 considered significant). High levels of methylated BCAT1 and IKZF1 were observed in tumour tissues (39.8%; 53.3%, respectively) compared with para-tumour tissues (3.2%; 0%, p<0.001). The increased IKZF1 methylation level was associated with a decrease in gene expression levels, with the tumours levels (0.030) significantly lower than the expression in para-tumour tissues (0.069; p<0.0001). No differences were observed in BCAT1 gene expression between tumour (0.077) and para-tumour (0.1247; p=0.7993). Compared to non-treated tissue, treatment reduced methylated BCAT1 and IKZF1 in tumour tissue (0.5%; 0.5%, p<0.01) without significantly affecting levels in para-tumour tissue (7.7%; 0.0%, p>0.05). The reduction in tumour methylation was accompanied by an increase in both BCAT1 (0.35) and IKZF1 (0.10) gene expression compared to non-treated levels (p<0.05). The levels of methylated BCAT1 and IKZF1 within the tumour was associated with the response to therapy, with significantly lower levels in tumours that had shrunk to 50% or less of the original size (both = 0%), compared to tumours with less size reduction (6.1% and 5.3%, p<0.05). Colorectal tumours, but not the adjacent non-tumour tissue, are highly methylated for BCAT1 and IKZF1. Methylation significantly reduced gene expression levels of IKZF1, but not BCAT1. Therapy reduced methylated BCAT1 and IKZF1 levels of the tumour and increased expression of BCAT1 and IKZF1. Levels of methylation may be able to be used as a marker for treatment success prior to surgical intervention.

Keywords: BCAT1, IKZF1, DNA methylation, colorecral cancer, neoadjuvant therapy

Subject: Medical Biotechnology thesis, Medicine thesis

Thesis type: Masters
Completed: 2016
School: School of Medicine
Supervisor: Dr. Erin Symonds